The number of colorectal cancer patients is currently continuing to increase rapidly each year in not only the U.S. and Europe, but in Japan as well, and is becoming one of the leading causes of cancer-related deaths. In Japan, this is thought to be due to the growing proliferation of a Western style diet consisting primarily of red meat among the Japanese people. More specifically, roughly 60,000 persons are diagnosed with colorectal cancer each year in Japan. In terms of the number of deaths by organ, colorectal cancer is ranked third after gastric cancer and lung cancer, and is predicted to continue to increase in the future. On the other hand, differing from other forms of cancer, colorectal cancer has a nearly 100% cure rate if treated soon after onset. Thus, it is of the utmost importance to include colorectal cancer in early cancer screening examinations, and in consideration thereof, research and development of testing methods for early discovery of colorectal cancer is proceeding at a rapid pace.
Methods such as barium enema examinations and colonoscopies are performed as testing methods for early discovery of colorectal cancer. Barium enema examinations consist of injecting barium into the large intestine and allowing it to adhere to the mucosal membranes of the large intestine, irradiating the intestine with X-rays to capture images of any surface irregularities, and then observing the surface. On the other hand, colonoscopy consists of observing the inside of the large intestine directly with an endoscope. Colonoscopy in particular enables high levels of sensitivity and specificity, while also offering the advantage of simultaneously allowing the excision of polyps and early forms of cancer.
However, in addition to be associated with high costs, these examinations place a considerable burden on the patient while also having the problem of being accompanied by complication risks. For example, barium enemas have risks associated with X-ray exposure and intestinal obstruction. In addition, colonoscopy is an invasive procedure for the subject since the endoscope is inserted directly into the large intestine. Moreover, the endoscopic procedures requires an experienced technician and the number of facilities were this examination can be performed is limited. Consequently, these examinations are not suitable for colorectal cancer examinations targeted at asymptomatic, healthy individuals as part of routine health examinations and the like.
In recent years, fecal occult blood tests have been widely performed as a non-invasive and inexpensive method for primary screening for colorectal cancer. The fecal occult blood test is a test for the presence of hemoglobin originating in erythrocytes contained in fecal matter, and is used as a method for indirectly predicting the presence of colorectal cancer. Factors behind the widespread use of the fecal occult blood test include stool samples being able to be collected and stored at room temperature eliminating the need for refrigerators, freezers and other special storage conditions, samples being able to be collected easily at home, and the test procedure being extremely simple. However, since the fecal occult blood test has low sensitivity of only about 25%, it has the problem of a high percentage of colorectal cancer being overlooked. Moreover, it also has a low positive predictive value, with the percentage of actual colorectal cancer patients among subjects judged to be positive in the fecal occult blood test being only 10% or less, thus resulting in a large number of false positives. Consequently, there is a strong need for the development of a new examination method offering higher reliability.
Attention is currently focusing on new examination methods that are suitable for routine health examinations by being non-invasive, simple and highly reliable for use in testing for the presence of cancer cells and cancer cell-derived genes in stool samples. Since these examination methods investigate the presence of cancer cells or cancer cell-derived genes directly, they are considered to be more reliable than the fecal occult blood test, which tests for the presence of blood from the digestive tract that occurs indirectly accompanying the onset of colorectal cancer.
In order to accurately detect cancer cells and the like in stool samples, it is important to efficiently recover cancer cell-derived nucleic acids from those stool samples. In particular, cancer cell-derived nucleic acids are only present in trace amounts in stool samples, and since stool samples also contain large amounts of digestive remnants and bacteria, nucleic acids are decomposed extremely easily. Consequently, in order to efficiently recover nucleic acids, and particularly nucleic acids derived from mammalian cells such as human cells, from stool samples, it is important to prevent decomposition of nucleic acids within the stool and prepare the stool sample so that it can be stored stably until the time of the actual testing procedure. An example of such a stool sample processing method consists of separating cancer cells that have dissociated from the large intestine or other constituent of the digestive tract from a collected stool sample. Separation of cancer cells from stool makes it possible to inhibit the effects of bacterial proteases, DNase, RNase and other degrading enzymes. An example of a method that has been disclosed for separating cells from stool consists of cooling the stool sample to a temperature below its gel freezing point, and collecting cells while maintaining at a temperature below the gel freezing point so that the stool substantially remains completely intact (see, for example, Patent Document 1). In addition, another method that has been disclosed consists of dispersing the stool sample in a transport medium containing a protease inhibitor, mucous dissolver and bactericide at a normal ambient temperature, followed by isolating the colorectal dissociated cells (see, for example, Patent Document 2).
On the other hand, numerous fixation methods, such as formalin fixation or alcohol fixation, have conventionally been employed to maintain the morphology of collected cells until the time of observation for the purpose histological and cytological observation of cell morphology. An example of a method that employs these fixation methods consists of a sample processing method that uses the following special cell solution preservative to enable mammalian cell samples to be stored for long periods of time or enable cells to be observed following storage (see, for example, Patent Document 3). This cell solution preservative includes an amount of water sufficient for colonizing mammalian cells, a miscible alcohol, an amount of anti-aggregation agent sufficient for preventing aggregation of mammalian cells in the solution, and a buffer for maintaining the pH of the solution within the range of 4 to 7 during the time the cells are stored.
In addition, a sample processing method that uses the storage solution described below is an example of a method that uses a storage solution allowing molecular analysis of proteins and nucleic acids present in cells following storage in addition to cell histological and cytological observations. Patent Document 4 discloses a sample processing method that uses a universal collection medium containing a buffer component, at least one alcohol component, a fixative component and at least one chemical agent that inhibits decomposition of at least one substance selected from the group consisting of RNA, DNA and protein, while Patent Document 5 discloses a sample processing method that uses a non-aqueous solution containing 5 to 20% polyethylene glycol and 80 to 95% methanol.
In addition, Patent Document 6 discloses a sample processing solution containing alcohol or ketone for stabilizing cells present in vaginal swabs and viral nucleic acids, and further containing a substance for precipitating or denaturing proteins along with a promoter for promoting injection into cells.
In addition, Patent Document 7 discloses a stool sample processing container that suspends an extremely small amount of collected stool sample on the order of about 0.03 g by mixing with a suspension containing a storage solution and the like for the purpose of fecal occult blood testing.